Macromolecular drugs such as polynucleotides, their mimics (peptide nucleic acids and phosphorodiamidate morpholino oligomers), and full-length proteins have great potential in the treatment of various diseases. Rapid development of such drugs is greatly facilitated by progress in biotechnology and by better understanding of the molecular mechanisms of pathogenesis. However, delivery of macromolecular drugs to their intracellular targets remains challenging, because along the way several permeability barriers have to be overcome, including a cell membrane that is generally impermeable to most macromolecules. Cationic cell-permeable peptides (CCPPs) such as nona arginine (R9) are a promising delivery vehicle for a wide range of potential macromolecular drugs and have been successfully used by many researchers to deliver different macromolecular cargo molecules both in vitro and in vivo. Despite significant progress in identification and design of new CCPPs, we still do not know mechanisms, by which higly charged CCPP cross cell membranes (plasma membrane or endosomal membrane) that are expected to present a non-permeable barrier enter cytosol and nucleus. In our earlier studies we found that at 37C CCPPs enter cells by endocytosis and are released into cytosol by mechanisms that may involve CCPP interaction with anionic lipids characteristic for late endosomal compartments. However the efficiency of endosomal escape mechanism is very low with most of the peptide (and associated cargo) trapped in endosomes and then degraded. In search for more efficient ways of drug delivery into the cytosol and nucleus we unexpectedly found that a rapid transfer of HeLa cells into cold medium (15C) in the presence of R9 (1-2&#956;M) dramatically boosts the efficiency of R9 entry into the cytosol and nucleus detected as nuclear labeling by rhodamine-tagged R9. The amount of the peptide entering an individual cell at 15C within 15 min was at least 50 times higher than the amount of the peptide entering a cell within 40 min at 37C by endocytosis. This temperature drop induced entry at relatively low peptide concentrations shares a number of similarities with the entry mechanism induced by high concentration of the peptide (> 10&#956;M, 37C). In particular, both of these entry pathways are inhibited by depletion of intracellular ATP and require a transient increase in intracellular calcium levels, indicating that both pathways depend on cell metabolism and intracellular signaling. Both entry of extracellular calcium and release of calcium from intracellular stores are required for temperature-drop-induced and high concentration-induced entry pathways. Our data indicate that interactions of CCPPs with cells activate intracellular signaling cascades that result in significant changes in plasma membrane permeability for highly cationic peptides. The phosphatidylserine-binding domain of lactadherin strongly inhibited peptide entry suggesting that non-endocytic cationic peptide entry involves Ca2+ dependent cell surface exposure of phosphatidylserine, a lipid normally residing only in the inner leaflet of the plasma membrane. To summarize, our work identifies a highly efficient intracellular-calcium-regulated pathway of oligo-arginine entry into cytosol and nucleus of adherent cells. Specific mechanisms by which cationic peptides activate intracellular signaling pathways and then cross the plasma membrane remain to be clarified. A better understanding of these mechanisms may enhance our knowledge of the properties of plasma membrane as well as guide development of future drug delivery vehicles.